Project Summary The goal of this investigation is to gain insight into the chemical mechanism of Fom3, a novel methyltransferase involved in the biosynthesis of the clinically useful antibiotic fosfomycin. Fom3 belongs to a unique class of cobalamin-containing Radical SAM (S-adenosyl-L-methionine) enzymes, which is involved in the biosynthesis of a number of pharmaceutically important compounds such as anti-microbial, anti-tumor, and anti-viral (including anti-HIV) agents. The mechanisms by which these enzymes carry out their unusual chemical transformations are unknown and may involve novel chemistry. The knowledge gained from this study has the potential to aid in the development of efficient strategies for the mass production of these compounds and may open new avenues for the rational design of pharmaceutical agents with new or improved biological function through the manipulation of these unprecedented reactions. To accomplish this goal, the products and stoichiometry of the reaction catalyzed by Fom3 will be determined using a combination of high-performance liquid chromatography (HPLC), mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). The stereo- and region-chemistry of the proposed hydrogen atom/proton transfers will then be established by determining the fate of deuterium atoms derived from stereospecifically-labeled substrate molecules using MS. Finally, the structures of key intermediates in the catalytic cycle of Fom3 will be characterized using UV/visible, electron paramagnetic resonance (EPR), and resonance Raman spectroscopies, in addition to transient kinetic analysis. Relevance The goal of this research is to study Fom3, an enzyme involved in the production of the antibiotic fosfomycin. Enzymes similar to Fom3 are involved in the production of a number of anti-cancer, anti- bacterial, and anti-viral (including anti-HIV) compounds. The way in which these enzymes carry out their function is unknown and the knowledge gained from this research has the potential to lead to the development of new Pharmaceuticals. [unreadable] [unreadable] [unreadable]